Development of a Recombinant Pichinde Virus-Vectored Vaccine Against Turkey Arthritis Reovirus and Its Immunological Response Characterization in Vaccinated Animals

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Article Development of a Recombinant Pichinde Virus-Vectored Vaccine against Turkey Arthritis Reovirus and Its Immunological Response Characterization in Vaccinated Animals

Pawan Kumar 1,2, Tamer A. Sharafeldin 1,3, Rahul Kumar 1,4 , Qinfeng Huang 5, Yuying Liang 5 , Sagar M. Goyal 1, Robert E. Porter 1, Hinh Ly 5,* and Sunil K. Mor 1,*

1 Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108, USA; [email protected] (P.K.); [email protected] (T.A.S.); [email protected] (R.K.); [email protected] (S.M.G.); [email protected] (R.E.P.) 2 Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, Haryana 125001, India 3 Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkia 44511, Egypt 4 Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Pandit Deen Dayal Upadhyaya Veterinary Science University and Cattle Research Institute, Mathura 281001, India 5 Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108, USA; [email protected] (Q.H.); [email protected] (Y.L.) * Correspondence: [email protected] (H.L.); [email protected] (S.K.M.); Tel.: +612-625-3358 (H.L.); +612-624-3698 (S.K.M.) Citation: Kumar, P.; Sharafeldin, T.A.; Kumar, R.; Huang, Q.; Liang, Y.; Abstract: Vaccination may be an effective way to reduce turkey arthritis reovirus (TARV)-induced Goyal, S.M.; Porter, R.E.; Ly, H.; Mor, lameness in turkey flocks. However, there are currently no commercial vaccines available against S.K. Development of a Recombinant TARV infection. Here, we describe the use of reverse genetics technology to generate a recombinant Pichinde Virus-Vectored Vaccine Pichinde virus (PICV) that expresses the Sigma C and/or Sigma B proteins of TARV as antigens. against Turkey Arthritis Reovirus and Nine recombinant PICV-based TARV vaccines were developed carrying the wild-type S1 (Sigma Its Immunological Response Characterization in Vaccinated C) and/or S3 (Sigma B) genes from three different TARV strains. In addition, three recombinant Animals. Pathogens 2021, 10, 197. PICV-based TARV vaccines were produced carrying codon-optimized S1 and/or S3 genes of a https://doi.org/10.3390/ TARV strain. The S1 and S3 genes and antigens were found to be expressed in virus-infected cells pathogens10020197 via reverse transcriptase polymerase chain reaction (RT-PCR) and the direct fluorescent antibody (DFA) technique, respectively. Turkey poults inoculated with the recombinant PICV-based TARV Academic Editor: Lawrence S. Young vaccine expressing the bivalent TARV S1 and S3 antigens developed high anti-TARV antibody titers, indicating the immunogenicity (and safety) of this vaccine. Future in vivo challenge studies using Received: 31 December 2020 a turkey reovirus infection model will determine the optimum dose and protective efficacy of this Accepted: 11 February 2021 recombinant virus-vectored candidate vaccine. Published: 13 February 2021

Keywords: Pichinde virus; recombinant vaccine; subunit vaccine; viral vectored vaccine; turkey Publisher’s Note: MDPI stays neutral arthritis reovirus; sigma C; sigma B with regard to jurisdictional claims in published maps and institutional afﬁl- iations.

1. Introduction Turkey arthritis reoviruses (TARVs) re-emerged in 2011 in Minnesota and other states in the United States (US) [1–3]. The viral genome consists of 10 segments of dsRNA grouped Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. into large (L1, L2, L3), medium (M1, M2, M3), and small (S1, S2, S3, S4) segments according This article is an open access article to their migration pattern on polyacrylamide gel electrophoresis [4,5]. The genome has distributed under the terms and 12 open reading frames (ORFs), which encode for eight structural and four nonstructural conditions of the Creative Commons proteins. The proteins encoded by L, M, and S genes are lambda (λ), mu (m), and sigma (σ), Attribution (CC BY) license (https:// respectively [5]. The S1 and S3 segments translate into σC (cell attachment) and σB (outer creativecommons.org/licenses/by/ capsid) proteins, respectively. The σC protein is a minor outer capsid protein of 326 amino 4.0/). acids, which has been identiﬁed as cell attachment protein responsible for virus entry into

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the host cell [6]. The σC protein mediates virus entry into the host cell via the Caveolin-1- mediated and dynamin-2-dependent endocytic pathway [7]. The σC protein is also known to induce apoptosis [8]. The C-terminal fragment of σC (residues 151–326) contains the receptor-binding globular domain [9]. The σC protein is the main immunogenic surface protein that possesses both type- and broad-specific epitopes, and it can elicit reovirus- specific neutralizing antibodies in the infected host [10]. The σB protein of 367 amino acids is a major component of the viral outer capsid that contains a group-specific neutralizing epitope [11]. Subunit vaccines for chicken arthritis reovirus (CARV) are available that contain either a partial fragment of σC protein [12] or a full-length σC protein alone [13] or in combination with σB protein [14]. The recombinant σC protein as a subunit vaccine has been used against duck reovirus [15]. Hence, σC and σB proteins have been commonly used in the development of subunit vaccines against avian reovirus infection in different avian species. Since TARVs are similar to CARVs in terms of their genomic structure, segment sizes, and open reading frames, the σC and σB of TARV should be considered in the development of mono- or multivalent virus vectored vaccines against TARV. TARV-infected turkeys display clinical lameness due to tenosynovitis and arthritis, resulting in huge economic losses mainly due to culling. No commercial vaccine is available to protect turkey flocks from the emerging TARV strains. Some turkey producers rely on the use of autogenous vaccines. However, the evolving nature of the virus creating new divergent strains poses a challenge for regular update of the vaccines. Additionally, there is no live vaccine available that can be used for priming before boosting breeder turkeys with injectable killed vaccines. Using a live and safe vectored vaccine to deliver TARV antigens is a reasonable alternative to live reovirus vaccines. Recently, a Pichinde virus (PICV) vector was developed that safely and effectively delivered the model antigens, the influenza viral hemagglutinin (HA), and nucleoprotein (NP) [16]. This arenavirus was first isolated from its natural host Oryzomys albigularis (rice rats) in the Pichinde valley of Colombia, South America [17]. Arenaviruses are enveloped RNA viruses with a bisegmented genome and are known to target dendritic cells and macrophages at the early stages of infection, making them a potentially powerful vaccine vector [18–21]. Using the reverse genetics technique, a live recombinant PICV (strain 18) with a trisegmented RNA genome (rP18tri) was developed to carry and express up to two foreign genes. One of the foreign genes could be the green fluorescent protein (GFP) that can be used to mark virus-infected cells in cell culture. rP18tri has been shown to be attenuated both in vitro and in vivo and has the ability to induce cell-mediated and humoral immune responses. For example, mice immunized with recombinant PICV–hemagglutinin (PICV– HA), expressing the modeled influenza hemagglutinin protein, developed a strong humoral response against HA that afforded complete protection against a lethal avian influenza virus infection [16]. The present study was undertaken to develop recombinant PICV vaccines expressing TARV antigenic Sigma C (σC) and/or Sigma B (σB) proteins. In addition, the vaccines were administered to turkey poults to determine their safety and efficacy. This is a “proof-of- concept” study for the development and recovery of recombinant PICV–TARV viruses, as well as for testing the safety and immunogenic properties of the turkey reovirus σC and σB protein(s) in vivo.

2. Materials and Methods 2.1. Cells and Viruses QT-35 cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) (Sigma- Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) and 50 µg/mL penicillin–streptomycin. Baby hamster kidney (BHK-21) cells, BSRT7-5 cells (BHK-21 cells stably expressing T7 RNA polymerase), and male leghorn chicken (LMH) cells were grown in Eagle’s minimal essential medium (MEM) (Sigma-Aldrich) that contained 10% FBS, 1 µg/mL gentamicin, and 50 µg/mL penicillin–streptomycin. Media for BSRT7-5 cells was Pathogens 2021, 10, 197 3 of 11

also supplemented with 1 µg/mL geneticin (Invitrogen-Life Technologies, Carlsbad, CA, USA). Three strains of turkey arthritis reovirus (SKM73, SKM95, and SKM121) isolated in QT-35 cells from tendons of lame turkeys were used. These viruses were selected on the basis of their pathogenicity and genomic characterization.

2.2. Pichinde Virus Plasmids Three plasmids were used: (i) pP18S1-GPC/MCS, which encodes the glycoprotein GPC and a multiple cloning site (MCS) to clone the gene of interest; (ii) pP18S2-MCS/NP, which encodes the nucleoprotein NP and an MCS; (iii) pP18L plasmid (the L plasmid), which expresses the full-length antigenomic strand of the rP18L segment under the control of the T7 promoter and does not contain any speciﬁc site to clone foreign genes [16].

2.3. Preparation of Vectors and Gene Inserts Genomic RNA was isolated from three TARV isolates (SKM73, SKM95, and SKM121). The full-length open reading frames (ORFs) of S1 and S3 genomic segments of these viruses were amplified. Additionally, the S1 and S3 ORF sequences of SKM121 were codon-optimized for expression in mammalian cells, commercially custom-synthesized, and cloned into a pUC vector (Genewiz, South Plainfield, NJ, USA). The primary sequences of these genes are provided in Supplementary Materials (Figure S1). The complementary DNA (cDNA) was synthesized using random primers and SuperScript™ IV First-Strand Synthesis (Thermo Fisher Scientific, Catalog#18091200, Waltham, MA, USA) and was PCR- amplified using specific cloning primers (Table1) and Phusion ®High-Fidelity PCR Master Mix with HF Buffer (NEB Catalog#M0531S, Ipswich, MA, USA). The NheI and Kozak sequences were added in the forward primer so that these features were included in the amplified product. Similarly, XhoI and sequence tags (FLAG tag in S1 and HA tag in S3 gene) were added to the reverse primer. The reaction conditions were as follows: 98 ◦C for 30 s (initial denaturation); five cycles of denaturation at 98 ◦C for 10 s, annealing at 66 ◦C for 30 s, and extension at 72 ◦C for 1 min; 30 cycles of denaturation at 98 ◦C for 10 s, annealing at 72 ◦C for 30 s, and extension at 72 ◦C for 1 min; final extension at 72 ◦C for 7 min and held at 4 ◦C. The PCR-amplified products (S1 and S3 genes of all three isolates) and the plasmids of the PICV (pP18S1-GPC/MCS and pP18S2-MCS /NP) were restriction enzyme-digested (NheI and XhoI, NEB) and gel-purified using the QIAquick gel extraction kit (Qiagen Catalog#28704, Germantown, MD, USA). The codon-optimized versions of ORFs were extracted from the pUC vector via restriction double digestion.

Table 1. List of primers used for full-length open reading frame (ORF) ampliﬁcation of S1 and S3 segments of turkey arthritis reovirus. The bold sequence part is speciﬁc and complementary for the turkey reovirus genes. The italicized sequence part is a tag sequence. The lowercase sequence part is a restriction enzyme (RE) site. The underlined sequence part is the Kozak sequence. TARV, turkey arthritis reovirus; F, forward; R, reverse; HA, hemagglutinin.

Expected PCR Position of Product Size Number of Primer on Scheme 5. Name Sequence (5’ to 3’) (Including RE Site Tag Bases Viral Template Primer (ORFs) Sequences) CGATgctagcGCCACCATGG 1 TARV-S1 F 36 1–20 NheI- CCGCTCTAACTCCGTC ATCGctcgagTTA 1031 bp CTTGTCGTCATCGTCTTTG- 2 TARV-S1 R 57 978–959 XhoI FLAG TAGTCGGTGTCGAT GCCCGTACGCA CGATgctagcGCCACCATGG 3 TARV-S3 F AGGTACGTGTGC- 41 1–25 NheI- CAAACTTTC ATCGctcgagTTAAGCGTAATC 1157 bp TGGAACATCG- 4 TARV-S3 R 64 1101–1078 XhoI HA TATGGGTACCAACCAC ACTCCATAAAAGTCAG Pathogens 2021, 10, 197 4 of 11

2.4. Cloning and Transfection The S1 and S3 gene inserts were ligated in the MCS region of plasmids pP18S2- MCS/NP and pP18S1-GPC/MCS, respectively, using 5 U/µL of T4 DNA ligase (Ther- moFisher Scientific, Catalog#EL0011). The ligation reaction mix was used to transform competent bacterial cells (DH5α) followed by selection using ampicillin antibiotic. All plasmids (plasmid pP18L, pP18S1-GPC/GFP, pP18S2-GFP/NP, and recombinant plasmids pP18S1-GPC/S3 and pP18S2-S1/NP) were isolated using the plasmid midi prep kit (Sigma- Aldrich). Recombinant plasmids were PCR-confirmed for reovirus genes and sequence- confirmed for correct orientation and reading frame. The recombinant plasmids were used to transfect BSRT7-5 cells in various combinations (Table2) using Lipofectamine ™ 3000 transfection reagent (ThermoFisher, Catalog#L3000008) following the manufacturer’s instruction with minor modifications. Briefly, BSRT7-5 cells were grown in six-well plates to 80% confluency. Four hours before transfection, the cells were washed, and fresh antibiotic- free medium was added. For transfection, 8 µL of P3000 reagent, 2 µg of L plasmid and 1 µg each of S1 and S2 plasmids were diluted in 250 µL of Opti-MEM (Invitrogen-Life Technologies) and incubated for 15 min at room temperature. In another tube, 10 µL of lipofectamine was diluted in 250 µL of Opti-MEM. Both mixtures were combined, followed by incubation at room temperature for 20 min to prepare DNA–lipid complexes. The cells were transfected with the resultant mixture, and MEM was changed after 4 h to remove the toxic lipofectamine. Then, at 48, 72, and 96 h post transfection, cell supernatants were collected and stored at −80 ◦C. Different monovalent and bivalent vector viruses were generated as detailed in Table2. The resultant viral recovery was confirmed by observing the green fluorescence of GFP in inoculated cell culture. The rescued virus was then grown in BHK-21 cells, and the GFP green fluorescence was observed. The expression of reovirus genes by the recombinant PICV vaccine virus was verified by RT-PCR assay.

Table 2. Pichinde virus (PICV) recombinant plasmids used to generate PICV-based TARV vaccines. GPC, glycoprotein; MCS, multiple cloning site; NP, nucleoprotein.

Serial Number of Recombinant Strains of Turkey Insert in Plasmid 1 Insert in Plasmid 2 Recombinant PICV-Based PICV-Based TARV Arthritis Reovirus pP18S1-GPC/MCS pP18S2-MCS/NP TARV Vaccines Vaccine Type 1 Monovalent GFP a S1 + wild-type SKM73 * 2 Monovalent S3 + wild-type GFP 3 Bivalent S3 wild-type S1 wild-type 4 Monovalent GFP S1 wild-type SKM95 * 5 Monovalent S3 wild-type GFP 6 Bivalent S3 wild-type S1 wild-type 7 Monovalent GFP S1 wild-type SKM121 * 8 Monovalent S3 wild-type GFP 9 Bivalent S3 wild-type S1 wild-type 10 Monovalent GFP S1 codon-optimized SKM121 * 11 Monovalent S3 codon-optimized GFP 12 Bivalent S3 codon-optimized S1 codon-optimized 13 Control GFP GFP a GFP = green ﬂuorescence protein. + S1 and S3 are genes inserted into PICV plasmids. * SKM73, SKM95, and SKM121 are designations of turkey arthritis reoviruses whose S1 and S3 genes were inserted into PICV plasmids.

2.5. Detection of Reovirus Antigenic Proteins The expression of σC and σB proteins by recombinant trisegmented PICV vaccine viruses in BHK-21 cells was veriﬁed using a direct ﬂuorescence antibody (DFA) assay. The BHK-21 cells were inoculated with the recombinant PICVs and at 96 h post infection (hpi); then, the cells were harvested, plated on 12-chamber slides, and dried for 2 h. Cells were Pathogens 2021, 10, 197 5 of 11

then fixed in acetone for 2 h followed by the addition of polyclonal Fluorescein isothio- cyanate (FITC)-conjugated anti-avian reovirus antibodies (National Veterinary Services ◦ Laboratory, Ames, IA, USA, Reagent#680-ADV). After incubation at 37 C in a CO2 incu- bator for 2 h, counterstaining was done using 0.1% Evan’s blue biological stain (EBBS). The slides were then mounted and examined under a fluorescent microscope to observe FITC green fluorescence, which was indicative of avian reovirus protein expression by the vaccine viruses.

2.6. Vaccination Experiment Four groups of turkey poults (ﬁve birds/group) were inoculated with 0.3 mL of the following recombinant PICVs containing codon-optimized gene segments of TARV- SKM121: PICV-based monovalent TARV-S1, monovalent TARV-S3, bivalent TARV-S1/S3, and PICV-control without any TARV segment insertion, having 3 × 105 plaque forming unit/mL (PFU/mL) via oral route at 1 week of age. Birds in all groups were given a booster dose (0.3 mL, 3 × 105 PFU/mL) with respective vaccine viruses at 3 weeks of age via intranasal (I/N) route. Blood samples were collected at 3 and 5 weeks of age. The birds were examined daily for any overt clinical signs or mortality. Birds displaying signs of severe illness were euthanized according to the guidelines laid by the Institutional Animal Care and Use Committee (IACUC) and research animal resources (RAR), University of Minnesota. At the end of the experiment, all birds were euthanized, and necropsy was done to detect the development of any gross lesions.

2.7. Serum Neutralization Assay Sera were separated from the collected blood samples and subjected to serum neutralization assay against virus strain TARV-SKM121. Brieﬂy, the heat-inactivated serum samples were fourfold serially diluted in a 96-well plate, and 25 µL of reovirus preparation (100 Median Tissue Culture Infectious Dose (TCID50)) was added to all wells except negative control wells. Subsequently, the virus–serum mixture was incubated at 37 ◦C for 1 h before adding it onto a freshly seeded primary hepatocellular carcinoma epithelial cell from a male leghorn chicken (LMH) (5 × 105 cells/well) with 10% fetal calf serum and incubated for 4–5 days. Virus-infected and uninfected cells were used as positive and negative controls, respectively. Virus controls, cell controls, and serum controls were included in each plate. The plates were observed daily for the appearance of cytopathic effects (CPEs). When a cytopathic effect was observed, the medium was removed, and the cells were stained with a 1% crystal violet solution prepared in 10% buffered formalin for 2–3 min, followed by two to three gentle washes with warm tap water. Plates were allowed to dry, and the titer was recorded as the reciprocal of the highest dilution of serum that inhibited at least 50% cell destruction. Serum neutralization titers among different groups were subjected to statistical analysis using nonparametric Kruskal–Wallis test followed by Mann–Whitney U test for testing statistical signiﬁcance at p < 0.05.

3. Results 3.1. Cloning of Reovirus Genes into PICV Plasmids The RT-PCR amplification of S1 (Sigma C) and S3 (Sigma B) ORFs yielded the expected product sizes of 1031 bp and 1157 bp, respectively (Figure1A). These products were gel-purified and cloned into pP18S2-MCS /NP and pP18S1-GPC/MCS, respectively. Restriction enzyme double digestion confirmed the presence of reovirus genes in recombinant PICV plasmids (Figure1B). Sanger sequencing confirmed the absence of unintended mutations in the cloned viral gene, as well as their correct reading frame and correct orientation in the vector backbones (data not shown). Pathogens 2021, 10, x FOR PEER REVIEW 6 of 12

2.7. Serum Neutralization Assay Sera were separated from the collected blood samples and subjected to serum neutralization assay against virus strain TARV-SKM121. Briefly, the heat-inactivated serum samples were fourfold serially diluted in a 96-well plate, and 25 µL of reovirus preparation (100 Median Tissue Culture Infectious Dose (TCID50)) was added to all wells except negative control wells. Subsequently, the virus–serum mixture was incubated at 37 °C for 1 h before adding it onto a freshly seeded primary hepatocellular carcinoma epithelial cell from a male leghorn chicken (LMH) (5 × 105 cells/well) with 10% fetal calf serum and incubated for 4–5 days. Virus-infected and uninfected cells were used as positive and negative controls, respectively. Virus controls, cell controls, and serum controls were included in each plate. The plates were observed daily for the appearance of cytopathic effects (CPEs). When a cytopathic effect was observed, the medium was removed, and the cells were stained with a 1% crystal violet solution prepared in 10% buffered formalin for 2–3 min, followed by two to three gentle washes with warm tap water. Plates were allowed to dry, and the titer was recorded as the reciprocal of the highest dilution of serum that inhibited at least 50% cell destruction. Serum neutralization titers among different groups were subjected to statistical analysis using nonparametric Kruskal–Wallis test followed by Mann–Whitney U test for testing statistical significance at p < 0.05.

3. Results 3.1. Cloning of Reovirus Genes into PICV Plasmids The RT-PCR amplification of S1 (Sigma C) and S3 (Sigma B) ORFs yielded the expected product sizes of 1031 bp and 1157 bp, respectively (Figure 1A). These products were gel-purified and cloned into pP18S2-MCS /NP and pP18S1-GPC/MCS, respectively. Restriction enzyme double digestion confirmed the presence of reovirus genes in recom- Pathogens 2021, 10, 197 binant PICV plasmids (Figure 1B). Sanger sequencing confirmed the absence of unin- 6 of 11 tended mutations in the cloned viral gene, as well as their correct reading frame and correct orientation in the vector backbones (data not shown).

Figure 1. Confirmation of turkey arthritis reovirus genes (S1 and S3) in the recombinant PICV vector Figure 1. Confirmation of turkey arthritis reovirus genes (S1 and S3) in the recombinant PICV vec- torby by restriction restriction enzyme enzyme (RE) (RE) double double digestion: digestion: (A) RT-PCR (A amplification) RT-PCR amplificationof full-length S1 (σC) of full-length S1 (σC) and Pathogens 2021, 10, x FOR PEER REVIEW and S3σ (σB) open reading frames of turkey arthritis reovirus. Lanes 1, 2, and 3: S1 gene of TARV 7 of 12 S3 ( B) open reading frames of turkey arthritis reovirus. Lanes 1, 2, and 3: S1 gene of TARV strains strainsSKM73, SKM73, SKM95, SKM95 and, and SKM121, SKM121, respectively respectively (1,031 (1,031 bp); M: bp); Marker; M: Marker; Lanes 4, 5, Lanes and 6: 4,S3 5,gene and 6: S3 gene of TARV of TARV strains SKM73, SKM95, and SKM121, respectively (1,157 bp); (B) Restriction enzyme (RE) strains SKM73, SKM95, and SKM121, respectively (1,157 bp); (B) Restriction enzyme (RE) double double digestiondigestion confirms confirms the presence the presence of S1 and of S1S3 andgenes S3 of genes TARV of in TARV recombinant in recombinant PICV plas- PICV plasmids. Lanes 1, mids. Lanes 1, 2,2, and and 3: 3: RE RE double double digestion of recombinantrecombinant pP18S2-S1/NPpP18S2-S1/NP plasmidplasmid yieldingyielding plasmid plas- backbone and mid backbone andcodon-optimized codon-optimized insert insert of 1031 of 1031 bp of bp S1 of ( σS1C); (σC); Lanes Lanes 4, 5, 4, and 5, and 6: RE 6: double RE double digestion di- of recombinant gestion of recombinantpP18S1-GPC/S3 pP18S1-GPC/S3 plasmid plasmid yielding yielding plasmid plasmid backbone backbone and codon-optimized and codon-optimized insert of 1157 bp of S3 insert of 1157 bp(σ ofB); S3 M: (σB Marker.); M: Marker.

3.2. Plasmid Transfection and Virus Rescue 3.2. Plasmid Transfection and Virus Rescue Viable recombinant PICVs were rescued successfully following transfection of Viable recombinant PICVs were rescued successfully following transfection of BSRT7- BSRT7-5 cells with the three plasmids in various combinations, as shown in Table 2. The 5 cells with the three plasmids in various combinations, as shown in Table2. The GFP GFP expression was observed at 48–72 h post transfection in cells transfected with at least expression was observed at 48–72 h post transfection in cells transfected with at least one GFP-containing plasmid (Figure 2A) (all monovalent vaccines in Table 2). The GFP- one GFP-containing plasmid (Figure2A) (all monovalent vaccines in Table2). The GFP- expressing fociexpressing increased fociin size increased over the in sizetime overcourse the of time transfection. course of The transfection. supernatants The supernatants were collectedwere from collected transfected from BSRT7 transfected-5 cells BSRT7-5and were cells used and to infect were usedBHK21 to infectcells. Strong BHK21 cells. Strong GFP expressionGFP was expression detected in was infected detected BHK21 in infected cells at 24 BHK21–48 hpi cells using at fluorescence 24–48 hpi using mi- fluorescence croscopy (Figuremicroscopy 2B), indicating (Figure the2B), rescue indicating of viable therescue PICV- ofbased viable TARV PICV-based vaccines TARV (viruses). vaccines (viruses). At every rescueAt attempt, every rescue we obtained attempt, infect we obtainedious viruses infectious at 48– viruses72 h post at transfection. 48–72 h post The transfection. The recombinant virusesrecombinant showed viruses minor showed GFP fluorescence minor GFP fluorescencein QT-35 and in LMH QT-35 cells and at LMH 96 hpi. cells at 96 hpi. As As expected, theexpected, bivalent the PICV bivalent-based PICV-based TARV vaccines TARV carrying vaccines carryingtwo TARV two genes TARV on genes both on both PICV PICV plasmidsplasmids did not didproduce not produce any green any fluorescence green fluorescence (Figure (Figure2C). 2C).

Figure 2. TransfectionFigure 2. Transfection and infection and of cells infection with plasmidsof cells with and plasmids recombinant and PICV-basedrecombinant TARV PICV vaccine-based TARV viruses, respectively. (A) GFP expressionvaccine inviruses, BSRT7-5 respectively cells was. observed(A) GFP expression under a ﬂuorescence in BSRT7-5 microscopecells was observed following under transfection a fluores- of cells with pP18S1-GPC/GFPcence microscope and pP18S2- following S1/NP transfection having a codon-optimized of cells with pP18S1 S1 gene-GPC/GFP insert, and as shownpP18S2 in- S1/NP Table having2, to generate a recombinanta PICV-basedcodon-optimized TARV S1 vaccine gene insert, number as shown 10. (B )in GFP Table expression 2, to generate in BHK-21 a recombinant cells infected PICV-based with supernatant of recombinantTARV monovalent vaccine PICV-based number 10. TARV (B) GFP vaccine expression number in 10 BHK at 96-21 hours cells post infected infection. with (supernatantC) BSRT7-5 cells of recom- transfected with binant monovalent PICV-based TARV vaccine number 10 at 96 hours post infection. (C) BSRT7-5 pP18S1-GPC/S3 and pP18S2- S1/NP having codon-optimized S1 and S3 gene inserts to generate a recombinant bivalent cells transfected with pP18S1-GPC/S3 and pP18S2- S1/NP having codon-optimized S1 and S3 gene PICV-based TARV vaccine (recombinant vector virus number 12 of Table2) that lacks the green ﬂuorescence. inserts to generate a recombinant bivalent PICV-based TARV vaccine (recombinant vector virus number 12 of Table 2) that lacks the green fluorescence.

3.3. Recombinant PICVs Expressing Reovirus Antigens Strong GFP expression by infected BHK21 cells indicated the successful rescue of recombinant PICV-based TRAV vaccines (viruses). The supernatant from infected BHK21 cells (passages P1, P2, and up to P3) was used to detect reovirus genes by RT-PCR. The results confirmed the presence of both viral genes in bivalent PICV-based TARV vaccines and either S1 or S3 gene in the monovalent vaccine viruses. The RT-PCR amplification indicated TARV antigen expression at the messenger RNA (mRNA) level, where the supernatant harvested at 48 h post infection showed significantly higher amplification than the supernatant harvested at 24 h post infection (Figure 3). To verify the expression of reovirus antigenic proteins (σC and σB) by the recombinant PICVs, we infected BHK21 cells with transfection supernatant and, then, at 96 hpi, conducted a direct fluorescence assay (DFA) using polyclonal FITC-conjugated anti-avian reovirus antibodies. The PICVs grown on BHK-21 showed varying degrees of fluorescence (Figure 4). The monovalent and bivalent PICV-based TARV vaccines that contained S1 and/or S3 showed fluorescence in BHK-21 cells (Figure 4A–E). Although we did not quantify the amount of fluorescence,

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3.3. Recombinant PICVs Expressing Reovirus Antigens Strong GFP expression by infected BHK21 cells indicated the successful rescue of recombinant PICV-based TRAV vaccines (viruses). The supernatant from infected BHK21 cells (passages P1, P2, and up to P3) was used to detect reovirus genes by RT-PCR. The results confirmed the presence of both viral genes in bivalent PICV-based TARV vaccines and either S1 or S3 gene in the monovalent vaccine viruses. The RT-PCR amplification indicated TARV antigen expression at the messenger RNA (mRNA) level, where the supernatant harvested at 48 h post infection showed significantly higher amplification than the supernatant harvested at 24 h post infection (Figure3). To verify the expression of reovirus antigenic proteins (σC and σB) by the recombinant PICVs, we infected BHK21 cells with Pathogens 2021, 10, x FOR PEER REVIEWtransfection supernatant and, then, at 96 hpi, conducted a direct fluorescence assay (DFA) 8 of 12

using polyclonal FITC-conjugated anti-avian reovirus antibodies. The PICVs grown on BHK-21 showed varying degrees of fluorescence (Figure4). The monovalent and bivalent PICV-based TARV vaccines that contained S1 and/or S3 showed fluorescence in BHK-21 PICVscells (Figure containing4A–E). SKM121 Although wegene did segments not quantify showed the amount a remarkably of fluorescence, higher PICVs degree of fluorescence,containing particularly SKM121 gene the segments bivalent showed PICV- abased remarkably TARV higher vaccine degree that of fluorescence, contained codon-opti- mizedparticularly S1 and the bivalentS3 segments PICV-based (Fig TARVure 4B). vaccine Minimal that contained fluorescence codon-optimized was observed S1 and in negative S3 segments (Figure4B). Minimal fluorescence was observed in negative controls (cells controlsthat contained (cells rescuedthat contained PICV without rescued any TARV PICV segment) without (Figure any 4TARVF). segment) (Figure 4F).

Figure 3. RT-PCR assay indicating the expression of turkey reovirus antigens at the messenger RNA Figure(mRNA) 3. levelRT-PCR by recombinant assay indicating PICV-based the TARV expression vaccines. of Lanes: turkey M, marker;reovirus A, recombinantantigens at PICV the messenger RNAcarrying (mRNA only GFP) level gene; by B, recombinant recombinant PICV PICV carrying-basedσ CTARV gene; C,vaccines. recombinant Lanes: PICV M carrying, markerσB; A, recombi- nantgene; PICV D, recombinant carrying PICVonly carryingGFP gene;σC and B, recombinantσB genes (after 24PICV h of infection);carrying E,σC recombinant gene; C, recombinant PICV PICV carryingcarrying σσBC andgene;σB D genes, recombinant (after 48 h of PICV infection). carrying σC and σB genes (after 24 h of infection); E, recombinant PICV carrying σC and σB genes (after 48 h of infection).

Figure 4. The recombinant PICV-based TARV vaccines expressing turkey arthritis reovirus antigens. Fluorescence in BHK-21 cells (green fluorescent cells) based on direct fluorescent antibody test indicates TARV protein expression. (A) SKM121 wild-type virus control; (B) PICV-based TARV vaccine carrying bivalent SKM121 codon-optimized S1 and S3 sequences; (C) PICV-based

Pathogens 2021, 10, x FOR PEER REVIEW 8 of 12

PICVs containing SKM121 gene segments showed a remarkably higher degree of fluorescence, particularly the bivalent PICV-based TARV vaccine that contained codon-optimized S1 and S3 segments (Figure 4B). Minimal fluorescence was observed in negative controls (cells that contained rescued PICV without any TARV segment) (Figure 4F).

Figure 3. RT-PCR assay indicating the expression of turkey reovirus antigens at the messenger RNA (mRNA) level by recombinant PICV-based TARV vaccines. Lanes: M, marker; A, recombi- Pathogensnant2021, 10PICV, 197 carrying only GFP gene; B, recombinant PICV carrying σC gene; C, recombinant PICV 8 of 11 carrying σB gene; D, recombinant PICV carrying σC and σB genes (after 24 h of infection); E, recombinant PICV carrying σC and σB genes (after 48 h of infection).

FigureFigure 4. The4. The recombinant recombinant PICV-based PICV-based TARV vaccinesTARV vaccines expressing expressing turkey arthritis turkey reovirus arthritis antigens. reovirus Fluorescence anti- ingens. BHK-21 Fluorescence cells (green ﬂuorescent in BHK-21 cells) cells based (green on direct fluorescent ﬂuorescent cells) antibody based test on indicatesdirect fluorescent TARV protein antibody expression. (A)test SKM121 indicates wild-type TARV virus protein control; expression (B) PICV-based. (A TARV) SKM121 vaccine wild carrying-type bivalent virus SKM121control; codon-optimized (B) PICV-based S1 and S3 sequences;TARV vaccine (C) PICV-based carrying TARV bivalent vaccine carryingSKM121 bivalent codon SKM121-optimized wild-type S1 and S1 and S3 S3sequences; sequences; ((DC)) PICV-based PICV-based TARV vaccine carrying monovalent SKM121 wild-type sequence; (E) PICV-based TARV vaccine carrying monovalent SKM121 codon-optimized sequence; (F) negative control.

3.4. Clinical Signs and Necropsy Birds inoculated with the PICV-based TARV vaccines (monovalent and bivalent) did not show any obvious clinical disease or illness during the study or any gross lesions at necropsy.

3.5. Serum Neutralization At 3 weeks of age, three of four sera from birds inoculated with the monovalent PICV- based TARV-S3 vaccine and four of five sera from birds inoculated with the bivalent PICV- based TARV S1/S3 vaccine showed serum neutralizing (SN) antibody titers of 64, which were significantly higher (p < 0.05) than the other two groups (Figure5). At 5 weeks of age, only one of three birds inoculated with monovalent PICV-based TARV-S1, monovalent PICV-based TARV-S3, and PICV-control had an SN antibody titer of 64, while all five sera from birds inoculated with the bivalent vaccine had high SN antibody titers ranging from 64 to 256, which were significantly higher (p < 0.05) than the other three groups (Figure5). Individual bird data show that there was a remarkable increase in the SN antibody titers of birds inoculated with the bivalent PICV-based TARV-S1/S3 vaccine at 5 weeks of age than the other groups (Figure5). Pathogens 2021, 10, x FOR PEER REVIEW 9 of 12

TARV vaccine carrying bivalent SKM121 wild-type S1 and S3 sequences ; (D) PICV-based TARV vaccine carrying monovalent SKM121 wild-type sequence; (E) PICV-based TARV vaccine carrying monovalent SKM121 codon-optimized sequence; (F) negative control.

3.5. Serum Neutralization At 3 weeks of age, three of four sera from birds inoculated with the monovalent PICV-based TARV-S3 vaccine and four of five sera from birds inoculated with the bivalent PICV-based TARV S1/S3 vaccine showed serum neutralizing (SN) antibody titers of 64, which were significantly higher (p < 0.05) than the other two groups (Figure 5). At 5 weeks of age, only one of three birds inoculated with monovalent PICV-based TARV-S1, monovalent PICV-based TARV-S3, and PICV-control had an SN antibody titer of 64, while all five sera from birds inoculated with the bivalent vaccine had high SN antibody titers ranging from 64 to 256, which were significantly higher (p < 0.05) than the other three groups Pathogens 2021, 10, 197(Figure 5). Individual bird data show that there was a remarkable increase in the SN anti- 9 of 11 body titers of birds inoculated with the bivalent PICV-based TARV-S1/S3 vaccine at 5 weeks of age than the other groups (Figure 5).

Figure 5. SN antibody titers of individual birds of different groups at 3 and 5 weeks of age in different groups. MonovalentS1, PICV-based TARV vaccine carrying monovalent PICV-SKM121 S1 (codon-optimized) sequence; MonovalentS3, PICV-based Figure 5. SN antibody titers of individual birds of different groups at 3 and 5 weeks of age in dif- TARV vaccine carrying monovalent PICV-SKM121 S3 (codon-optimized) sequence; Bivalent S1/S3, PICV-based TARV ferent groups. MonovalentS1, PICV-based TARV vaccine carrying monovalent PICV-SKM121 S1 vaccine carrying(codon bivalent-optimized) PICV-SKM121 sequence; S1/S3 MonovalentS3 (codon-optimized), PICV-based sequences; TARV vaccine Vaccine carrying Control, PICVmonovalent vaccine with no insert (control). A281PICV to-SKM121 A300 represent S3 (codon tags-optimized) on individual sequence; birds. Bivalent The dotted S1/S3 lines, PICV represent-based TARV the mean vaccine values carrying at 3 weeks old and the solidbiva lineslent represent PICV-SKM121 the mean S1/S3 values (codon at-optimized) 5 weeks old. sequences; The values Vaccine of Monovalent Control, PICV S3 and vaccine Bivalent with S1/S3 groups were significantlyno insert higher (control). than theA281 other to A300 two represent groups at tags 3 weeks on individual old (+), and bird thes. valuesThe dotted of the lines Bivalent represent S1/S3 the group were significantlymean higher values than theat 3 otherweek threes old and groups the atsolid 5 weeks lines oldrepresent (*) using the the mean Mann–Whitney values at 5 week U tests old. at p The< 0.05. values of Monovalent S3 and Bivalent S1/S3 groups were significantly higher than the other two groups at 3 weeks old (+), and the values of the Bivalent S1/S3 group were significantly higher than the other three4. Discussion groups at 5 weeks old (*) using the Mann–Whitney U test at p < 0.05. Using PICV as a vector to express TARV antigenic proteins presents an attractive 4. Discussion alternative to the use of live attenuated TARV vaccines that may result in the emergence of Using PICVmutant as a TARVvector strains. to express Recombinant TARV antigenic PICV expressing proteins thepresents hemagglutinin an attractive and nucleoprotein alternative to thegenes use of of influenza live attenuated initiated TARV humoral vaccines and cell-mediatedthat may result immune in the emergence responses and provided full protection against lethal influenza in mice [16]. Several subunit vaccines reported against chicken reovirus and duck reovirus involve expression of the σC protein alone or in combination with σB proteins [12–15]. The purpose of this study was to develop recombinant PICV-based TARV vaccines that carry S1 and/or S3 genes of turkey arthritis reoviruses. We successfully recovered recombinant PICV after insertion of TARV genomic segments in the PICV plasmids, detected the inserted genes, and confirmed the expression of recombinant antigenic proteins. A total of 12 different recombinant PICVs were developed. We used the wild-type genes from three different TARV isolates in addition to codon-optimized genes from one TARV isolate in an effort to find an optimum TARV candidate to be used in developing a vaccine. The recovered PICV recombinants grew well in BHK-21 cells but showed minimal growth on QT-35 and LMH cells as determined by the expression of GFP in recombinant PICV that contained each of the TARV genes and the GFP gene. No GFP gene was inserted in the bivalent recombinant PICV (containing both S1 and S3 genes of TARV) and, hence, they could not be subjected to the green fluorescence test. We assume that these viruses grew the same way as the monovalent viruses because their plasmids and transfection were done under the same conditions. The detection of the inserted genes and the protein expression was helpful in determining the successful rescue and growth of the bivalent PICV-based TARV vaccines. The use of RT-PCR to detect the inserted TARV genome in recovered viruses helped in determining the success of recovering recombinant PICV-based TARV vaccines that contained TARV S1 and/or S3 genes. Pathogens 2021, 10, 197 10 of 11

To confirm the expression of reoviral antigenic proteins by the recombinant PICV- based TARV vaccines, we used the direct fluorescent antibody technique (DFA) using FITC-conjugated anti-avian reovirus antibodies. The FA procedure included dehydration and long fixation with acetone. These two steps helped eliminate GFP fluorescence and increased permeabilization of the cell membrane. The permeabilization of the cell membrane enabled the entrance of the FITC-conjugated antibody to reach the intracellular recombinant viral proteins. Although we did not have a quantitative method to measure the amount of the expressed protein, we could subjectively observe that the fluorescence produced by viruses recombined with SKM121 genes was more than that produced by SKM73 and SKM95. The bivalent PICV-based TARV vaccines that contained codon-optimized S1 and S3 of SKM121 showed the best fluorescence, indicating the growth of these viruses, which did not have any GFP fluorescence after transfection. Since codon-optimized PICV-based TARV SKM121 vaccines showed the best expression of TARV S1 and/or S3 proteins, we used these vaccines for a pilot in vivo experiment. PICV without an insertion of TARV gene was used as a control. The in vivo experiment was to determine the safety and humoral response generated by the recombinant vaccines in turkey poults. Turkey poults were vaccinated with the same dose of vaccine (0.3 mL, 3 × 105 PFU/mL) via oral and intranasal route using a prime-boost strategy. Poults were primed at 1 week of age because birds are susceptible to avian reovirus infection in the early days of their life [22,23]; hence, vaccination strategies are designed to provide passive immunity from maternal antibodies by vaccinating breeders or by actively immunizing young bids with a live vaccine [24]. Priming at 1 week of age was also considered to avoid vaccination shock and poor intestinal immunity in day old birds [24]. Booster dose was given intranasally at 3 weeks of age, targeting the coarse spray administration with the same vaccine as previously described [25]. The booster vaccination was done after 2 weeks of priming because the recombinant PICV-based vaccine needs 2–3 weeks to provoke the best immune responses. Immunogenicity of the codon-optimized bivalent PICV-based TARV SKM121 vaccine was demonstrated by the production of high SN antibody titers. In future in vivo experiments, we plan to further characterize the effect of the dose of the recombinant PICV-based TARV vaccines on antibody titer and response to reovirus challenge in turkeys, as well as to study the efficacy of protection of the PICV-based TARV vaccines against heterologous and homologous challenge viruses.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-0817/ 10/2/197/s1, Figure S1: Codon optimized and wild-type sequences of TARV sigma C and sigma B genes. Author Contributions: Conceptualization, S.K.M., S.M.G., and H.L.; methodology, P.K., S.K.M., and H.L.; validation, P.K., T.A.S., and R.K.; formal data analysis, P.K., T.A.S., H.L., S.K.M., and R.K.; investigation, P.K., T.A.S., R.K., R.E.P., and Q.H.; resources, S.K.M., R.E.P., and H.L.; data curation, P.K., T.A.S., and R.K.; writing—original draft preparation, P.K. and T.A.S.; writing—review and editing, P.K., T.A.S., R.K., R.E.P., Y.L., S.K.M., S.M.G., and H.L.; visualization, P.K.; research supervision, S.K.M., S.M.G., H.L., and Y.L.; project administration, S.K.M., S.M.G., H.L., and Y.L.; funding acquisition, S.K.M. and H.L. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded in part by research grants from the Minnesota Agricultural Experi- ment Station’s Rapid Agricultural Response Funds to S.K.M. and H.L. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are openly available in FigShare at 10.6084/m9.figshare.13980386. Conflicts of Interest: The authors declare no conflict of interest. Pathogens 2021, 10, 197 11 of 11

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